The long-term goal of our research is to develop new improved treatments for drug-resistant epilepsies. The ketogenic diet (KD), an extremely high fat diet, is one of the few effective treatments for drug-resistant epilepsies in children, but it is very difficult to maintain. The anticonvulsant mechanisms of the KD are still unknown. One main characteristic of the diet is that it increases the blood levels of ketones, including D-[unreadable]-hydroxybutyrate (D-BHB), acetoacetate, its degradation product acetone, and potentially L-[unreadable]-hydroxybutyrate (L-BHB). Our hypothesis is that chronic elevation of D-BHB and/or acetone in the brain is sufficient for the KD's anticonvulsant effect. If our experiments show that our hypothesis is correct, it would lead to the development of new dietary forms of ketones for improved therapy of drug-resistant epilepsy. We propose two aims. Our first aim is to determine the anticonvulsant effect of the KD in mice and then to quantify the levels of D-BHB, L-BHB, acetoacetate, and acetone in brain and blood of KD-fed mice, which are protected against seizures. Brain and blood samples will be reacted with borodeuteride and the levels of all major ketones, namely D-BHB, acetoacetate, L-BHB and acetone, will be quantified by gas chromatography-mass spectroscopy at the Mouse Metabolic Phenotyping Center at Case Western University. To establish the composition of the brain extracellular fluid in KD-vo control diet fed mice, we will use in vivo microdialysis to sample and then quantify the concentrations of D-BHB, acetoacetate, glucose, and lactate. Our second aim is to identify the effect of chronic vs. acute delivery of D-BHB and acetone in mouse seizure models in comparison to the KD. We hypothesize that chronically high D-BHB and/or acetone levels in the brain are anticonvulsant. To test this hypothesis we will compare seizure thresholds in the 6 Hz model of mice a) on control diet, b) on KD vs. those with chronic and acute elevation of brain ketones induced by feeding c) triolide, the cyclic trimer of D-BHB , and d) trioctanoin, the triglyceride of octanoate, and e) with chronic and acute elevation of brain acetone induced by supplying acetone in the drinking water or acute acetone injection (i.p.). We expect to find that chronic elevation of brain D-BHB to levels found during KD-feeding is anticonvulsant, potentially by degrading to acetone. The completion of this study will add to the understanding of the role of high ketone levels in the anticonvulsant mechanism(s) of the KD. Our data may lead to an improved therapy of epilepsy by directly feeding dietary ketone formulations, which lack sodium and fat, such as triolide. Finally, our data on the dietary use of triolide or trioctanoin as ketogenic therapies will provide important insight into for the treatment of other disorders, in which ketones and the KD were found to be protective, including stroke, Parkinson's and Alzheimer's disease and brain injury. PUBLIC HEALTH RELEVANCE: Project Narrative Knowledge gained from this proposal will provide new insights into the anticonvulsant mechanism of the ketogenic diet and how seizures can be prevented. A sodium- and acid- free dietary form of ketones will be developed, which may be used to treat pharmacoresistant epilepsy and a broad range of conditions associated with neurodegeneration, such as stroke, Alzheimer's Disease, Parkinson's Disease, and brain injury. This substance may also be useful to treat metabolic disorders, including insulin resistance, and other diseases resulting from free radical damage or hypoxia.